Acoustic separation of particles for bioprocessing

ABSTRACT

A method for separating particles in a biofluid includes pretreating the biofluid by introducing an additive, flowing the pretreated biofluid through a microfluidic separation channel, and applying acoustic energy to the microfluidic separation channel. A system for microfluidic separation, capable of separating target particles from non-target particles in a biofluid includes at least one microfluidic separation channel, a source of biofluid, a source of additive, and at least one acoustic transducer coupled to the microfluidic separation channel. A kit for microfluidic particle separation includes a microfluidic separation channel connected to an acoustic transducer, a source of an additive, and instructions for use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to U.S.Provisional Patent Application No. 62/492,044 titled “ACOUSTICSEPARATION OF PARTICLES FOR BIOPROCESSING” filed on Apr. 28, 2017, whichis hereby incorporated by reference in its entirety for all purposes.

FIELD OF TECHNOLOGY

Aspects and embodiments disclosed herein relate to systems and methodsfor the separation of particles in a biofluid. In particular, aspectsand embodiments disclosed herein relate to systems and methods for theseparation of target particles in a biofluid from non-target particlesin the biofluid.

SUMMARY

In accordance with an aspect, there is provided a method of separatingtarget particles from non-target particles in a biofluid. Specifically,there is provided a method for separating target particles that wereintroduced into the biofluid to provide a therapeutic treatment to atleast one component of the biofluid. The method may comprise pretreatingthe biofluid, flowing the pretreated biofluid into an inlet of amicrofluidic separation channel, and applying acoustic energy to themicrofluidic separation channel to accumulate target particles within aprimary stream along the separation channel and accumulate non-targetparticles within a secondary stream along the separation channel. Insome embodiments, pretreating the biofluid comprises introducing anadditive into the biofluid to alter at least one of size of the targetparticles, size of the non-target particles, compressibility of thebiofluid, compressibility of the target particles, compressibility ofthe non-target particles, aggregation potential of the target particles,and aggregation potential of the non-target particles. The method mayfurther comprise introducing an additive to alter at least one ofdensity of the biofluid, density of the target particles, and density ofthe non-target particles. In some embodiments, the acoustic energy maybe applied transverse to a direction of the fluid flow through theseparation channel.

In some embodiments, the method further comprises selecting the biofluidfrom blood buffy coat, leukapheresis product, peripheral blood, wholeblood, lymph fluid, synovial fluid, spinal fluid, bone marrow, ascitiesfluid, and combinations or subcomponents thereof.

According to some embodiments, the method further comprises selectingthe target particles to be synthetic particles selected from the groupconsisting of carrier particles, capture particles, enrichmentparticles, delivery particles, subclasses thereof, and combinationsthereof. In some embodiments the target particles were introduced intothe biofluid to provide a therapeutic treatment to at least onecomponent of the biofluid. For instance, the therapeutic treatmentprovided by the target particle prior to pretreating the biofluid may beselected from the group consisting of delivering a therapeutic moiety,capturing a therapeutic moiety, capturing a toxin, capturing a celltype, capturing a synthetic particle, culturing a cell type, andcombinations thereof.

In accordance with one embodiment, there is provided a method ofseparating synthetic particles from non-target particles. According tocertain embodiments, there is provided a method of separating carrierparticles from non-target particles. According to some embodiments themethod comprises selecting the target particles to be cell culturecarrier particles.

The biofluid may be collected from a donor subject. The secondary streammay be post-treated and delivered to a recipient subject. In someembodiments, the recipient subject is the same as the donor subject. Themethod may be performed in line such that the biofluid is collected froma subject, target particles are separated from non-target particles inthe biofluid by a method as described herein, a fluid depleted of targetparticles may be post-treated, and the post-treated fluid may bedelivered back to the subject. In some embodiments, the donor subjectand the recipient subject are not the same subject. The fluid depletedof target particles may be collected and stored for delivery to therecipient subject at a later time.

The method may further comprise flowing the fluid comprising the targetparticles through microfluidic separation channels arranged in seriesand applying acoustic energy to each separation channel. In someembodiments, the biofluid comprising target particles may be flowedthrough a first microfluidic separation channel to produce a primarystream enriched in target particles and a secondary stream depleted oftarget particles. The secondary stream may then be flowed through asecond microfluidic separation channel to produce a second or subsequentsecondary stream having a higher purity of non-target particles.

In some embodiments, the biofluid comprises non-target particles boundto the target particles. The method may further comprise treating thebiofluid to unbind the non-target particles from the target particlesprior to pretreating the biofluid for separation.

According to certain embodiments, the method further comprises flowing asecond fluid adjacent to the biofluid into an inlet of the microfluidicseparation channel. The biofluid and the second fluid may flow throughthe separation channel in substantially parallel and substantiallylaminar form.

In some embodiments the non-target particles comprise at least one oflive cells, frozen cells, preserved cells, or cells grown in a cellculture. In some embodiments, the microfluidic separation channel isformed of a thermoplastic material. The microfluidic separation channelmay be disposable.

In accordance with another aspect, there is provided a system formicrofluidic particle separation. The system may be configured toseparate target particles from non-target particles in a biofluid.Specifically, the system may be configured to separate target particlesintroduced into a biofluid to provide a therapeutic treatment to atleast one component of the biofluid. In some embodiments, the systemcomprises at least one microfluidic separation channel comprising atleast one inlet, a first outlet, and a second outlet, a source ofbiofluid in fluid communication with the microfluidic separationchannel, a source of additive in fluid communication with the source ofthe biofluid, configured to introduce at least one additive into thebiofluid, and at least one acoustic transducer coupled to a wall of themicrofluidic separation channel. The acoustic transducer may bepositioned to apply a standing acoustic wave transverse to themicrofluidic separation channel. Systems that comprise more than onemicrofluidic separation channel may comprise one acoustic transducercoupled to each microfluidic separation channel or one or more acoustictransducers coupled to a collection of microfluidic separation channels.

In some embodiments, the system comprises at least two microfluidicseparation channels. The at least two microfluidic separation channelsmay be arranged in a parallel arrangement. The system may furthercomprise a manifold configured to distribute biofluid to the at leasttwo microfluidic separation channels. In some embodiments, the manifoldis configured to distribute the biofluid in response to the inputbiofluid load on the system. The system may further comprise a sensorconfigured to measure an input biofluid load on the system. The sensormay be in electrical communication with the manifold, such that themanifold may distribute the biofluid to the microfluidic separationchannels in response to the measurement of the input biofluid load.

In some embodiments, the system further comprises at least one sensorconfigured to measure at least one of density of the biofluid,hematocrit (HCT %) of the biofluid, concentration of target particles,or concentration of non-target particles in the biofluid. The system mayfurther comprise a control module in electrical communication with thebiofluid sensor. The control module may be in electrical communicationwith the source of additive, and configured to introduce a predeterminedvolume of the additive into the biofluid in response to the measurementof density of the biofluid or concentration of target particles ornon-target particles. In certain embodiments the predetermined volume ofthe additive is determined to alter or regulate the biofluid to have adesired density, HCT %, or concentration of target particles ornon-target particles.

According to certain embodiments, the system further comprises at leastone sensor configured to measure a parameter of an output suspension.The sensors may measure HCT %, concentration of target particles, orconcentration of non-target particles in the primary or secondarystream. The system may further comprise a control module in electricalcommunication with the output suspension sensor. The control module maybe in electrical communication with the acoustic transducer, andconfigured to alter or regulate at least one input parameter of theacoustic transducer. For instance, the control module may alter orregulate the power, voltage, or frequency delivered to the acoustictransducer in response to a measurement of a parameter of the outputsuspension. For instance, the control module may be configured to act inresponse to a measurement of HCT %, concentration of target particles,or concentration of non-target particles in the output suspension. Thecontrol module in communication with the output suspension sensor may bethe same or different from the control module in communication with thebiofluid sensor.

The system may further comprise a source of a second fluid in fluidcommunication with the at least one inlet of the at least onemicrofluidic separation channel. The source of the second fluid may beconfigured to introduce the second fluid into the biofluid. In someembodiments, the biofluid and the second fluid flow in substantiallyparallel, substantially laminar flow.

In some embodiments, the system may further comprise a first and secondcollection channel in fluid communication with the at least one outletof the microfluidic separation channel. A collection vessel may be influid communication with the first or second collection channel. Thesystem may further be connectable to an intraluminal line. For instance,the system may be connectable to an intraluminal line configured toextract biofluid from a donor subject and deliver it to the source ofthe biofluid for processing. The system may be connectable to anintraluminal line configured to deliver an output suspension, forexample target particle depleted fluid, to the recipient subject.

The system may further comprise a source of a target particle. Thesource of the target particle may be configured to deliver targetparticles to the biofluid for therapeutic treatment to the at least onecomponent of the biofluid.

In some embodiments, the system further comprises a target particleprocessing chamber. The target particle processing chamber may beconfigured to unbind non-target particles from target particles prior toseparation. In some embodiments, the target particle processing chamberis fluidly connected to a source of a treatment fluid configured tofacilitate detachment of the non-target particles from the targetparticles.

According to certain embodiments, the system further comprises a recycleline. The recycle line may be configured to deliver output suspensionback to the source of the biofluid for a second pass separation. Theoutput suspension may be target particle enriched fluid or targetparticle depleted fluid. The recycle line may be configured to recycletarget particle depleted fluid from the second outlet to the source ofthe biofluid. In some embodiments, the system comprises more than onemicrofluidic separation channel arranged in series.

In accordance with another aspect, there is provided a kit forseparation of target particles from non-target particles. The kit maycomprise at least one microfluidic separation channel connected to anacoustic transducer, a source of an additive fluidly connectable to theat least one inlet of the microfluidic separation channel, andinstructions for use.

The kit may include instructions to collect a biofluid, introduce targetparticles into the biofluid to provide a therapeutic treatment to atleast one component of the biofluid, pretreat the biofluid byintroducing a predetermined volume of additive into the source of thebiofluid, flow the pretreated biofluid through the microfluidicseparation channel, and apply acoustic energy to the separation channel.In some embodiments, the kit provides instructions to introduce theadditive to alter or regulate the density of the biofluid orconcentration of the target particles or non-target particles.

According to certain embodiments, the kit may further comprise acollection channel, a collection vessel, a manifold system, a sensor, acontrol module, or an intraluminal line.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a schematic drawing of a microfluidic separation channel,according to one embodiment;

FIG. 2 is a schematic drawing of an alternate microfluidic separationchannel, according to another embodiment;

FIG. 3 is a micrograph of a microfluidic separation channel coupled toan acoustic transducer that is turned off;

FIG. 4 is a micrograph of a microfluidic separation channel coupled toan acoustic transducer that is turned on;

FIG. 5 is a schematic drawing of a system for microfluidic particleseparation, according to one embodiment;

FIG. 6 is a schematic drawing of an alternate system for microfluidicparticle separation, according to another embodiment;

FIG. 7 is a schematic drawing of an alternate system for microfluidicparticle separation, according to another embodiment; and FIG. 8 is aschematic drawing of an alternate microfluidic separation channel,according to another embodiment.

DETAILED DESCRIPTION

In the fields of bioprocessing and cell therapy emerging medicaltechniques may involve use or introduction of a particle into a biofluidsample for therapeutic treatment. The therapeutically treated orengineered biofluid sample may be introduced into a patient to medicaltreatment. In many applications, the particle may be a foreign particlethat is not desirably introduced into a patient.

For instance, cell therapy and bioprocessing methods may involveextraction of blood or tissue from a patient followed by purification ofa particular cell type from a sample. In some applications, theparticular cell type is prepared for treatment or manipulated before itis re-injected into a patient. In certain applications, the particularcell type is identified with a capture particle. After processing, thecell type may be detached from the capture particle, leaving asuspension of capture particles and cells in the fluid. Aspects andembodiments disclosed herein relate to separation of used captureparticles from a liquid suspension of mixed cells and particles. Inparticular, one example application is the separation of synthetic cellcapture particles from a blood sample. For instance, synthetic captureparticles with surface CD3+, CD4+, or CD8+antibodies designed toselectively bind T-lymphocytes from a blood sample.

In another exemplary application, magnetic processing of cells can behighly selective, but depends on the attachment of paramagnetic captureparticles to cells using affinity ligands, such as antibodies. Theparticles may pose a safety risk if injected into a patient.Accordingly, the magnetic particles must be removed from a finaltherapeutic product.

Aspects and embodiments disclosed herein may relate to methods andsystems for use in processing of cells for cell therapy. Many other usesof some of the embodiments described herein could also be envisioned, inparticular wherever a foreign particle is desired to be introduced intoa biofluid to provide a therapeutic treatment, and then collected fromthe biofluid sample after treatment. Some non-limiting examples includethe diagnostic or environmental monitoring assays, tissue engineering,in vitro models, and biomanufacturing systems, such as for energyapplications.

Formerly washing of biofluid samples to remove foreign particles hasbeen performed by one or more batch centrifugations, continuouscentrifugations, magnetic separation, or combinations thereof.Centrifugation is only able to separate particles by density, limitingits ability to separate particles from similarly dense cells. Additionof a density medium may improve particle separation, but only in smallbatch procedures requiring technically trained operators.

Membrane based filtration may be partially successful in removingforeign particles from a suspension sample, but difficulties persist.Generally, membrane based filtration provides only size exclusion ofparticles. Foreign capture particles may be essentially similar in sizeto desired cells, creating difficulty in selectively separation foreignparticles from a cell suspension.

Aspects and embodiments disclosed herein may be advantageous overprevious sample purification technologies because, for example, in someembodiments the removal of particles can be performed continuously, insome embodiments, the systems and methods provide separation by bothsize and density to further enhance particle separation, in someembodiments the separation processes may be readily scaled to small orlarge sample volumes, in some embodiments, a high degree of purificationcan be achieved with the addition of safely injectable, physiologicallyacceptable additives.

One non-limiting example application of the methods and systemsdisclosed herein is large scale bioprocessing. In some bioprocessescells, such as mesenchymal stem cells, may be cultured on carrierparticles. The carrier particles may be suspended in a medium andprovide a substrate needed for cell development. Before the culturedcells may be used in therapies or research, they must be detached fromthe carrier particles, for example by biochemical separation. Thedetached carrier particles must be removed from the suspension toharvest the final cell product.

The aspects and embodiments disclosed herein may improve methods forselectively separating target particles from a biofluid, and may alsohave applications in other steps in the process such as purification ofsamples after transduction.

Acoustic separation, also referred to as acoustophoresis, may be used toisolate or enrich desired cells as part of a bioprocessing workflow.Acoustic separation of particles in a biofluid has been described in,for example, U.S. Patent Application Publication Nos. 2016-0030660,2016-0008532, and 2013-0048565, and in U.S. Pat. No. 9,504,780, each ofwhich is herein incorporated by reference in their entirety. The aspectsand embodiments disclosed herein provide separation of a target particlefrom a liquid suspension of mixed cell types including other non-targetparticle types. More specifically, the aspects and embodiments disclosedherein provide improved selective separation of target particles from abiofluid suspension by introducing a physiologically acceptable additiveto alter one or more physical properties of the fluid, target particle,or non-target particles.

In acoustic separation, a mixed suspension may flow through a duct thatis oscillated at ultrasonic frequencies by an external mechanicaloscillator. The duct may form a resonant cavity, for instance so thatultrasonic pressure waves are generated and contact the flow across theduct. For example, the ultrasonic waves may be generated at an anglerelative to the flow. Ultrasonic waves may be generated in a directionsubstantially transverse to the flow. Cells or other particles in thesuspension may experience a force from the pressure waves and migrate tonodes in the resulting pressure field. The rate at which particlesmigrate generally depends on their size, density, and compressibility.Separation may be facilitated, for example, by larger and more denseparticles migrating to a pressure node, with smaller or neutrallybuoyant particles migrating slowly, not migrating (substantially stayingon axis), or migrating to anti-nodes. For instance, in a typicalconfiguration separation process, the pressure node is established alongthe axis of the duct and certain particles may move to this pressurenode axis and flow in a concentrated stream along it, while otherparticles may remain disperse or move to a pressure anti-node axis.

Referring again to the example application of large scale bioprocessing,carrier particles may be preferentially extracted from cell culturesamples. The separation may involve altering a property of the cellculture suspension, of the carrier particles, or of a certain class ofcells within the suspension, such that the carrier particles are lesssusceptible to acoustic energy than, for example the stem cells andother classes of cells. Therefore, when a biofluid, for example a cellculture suspension, is passed through an acoustic separator, carrierparticles may remain in a side stream with greater abundance than thedesired cells. The side stream may be discarded or collected forprocessing and the center stream may be collected. Conversely, theseparation may involve altering a property of the carrier particles tomake them more susceptible to the acoustic energy. For instance, ironnanoparticles may be added to a polymeric carrier particle. In otherembodiments, the carrier particle may comprise a polymer of higherdensity or lower compressibility than the class of cells cultured.

In certain embodiments, the carrier particle may be designed orengineered to provide a modified carrier particle having properties thatfurther enhance separation from target cells. For instance, the particlematerial may be modified or engineered to facilitate separation from thetarget cells, or the particle may comprise a filler that is designed,modified, or engineered to have specific properties that facilitateseparation from the target cells.

In accordance with an aspect, there is provided a method of separatingtarget particles from non-target particles in a biofluid. Morespecifically, there is provided a method for selective, differentialseparation of a desired particle from a biofluid comprising a suspensionof mixed cell types and other particles. Target particles which may beselectively separated from the mixed cell types and other particles inthe suspension include synthetic particles, carrier particles, captureparticles, enrichment particles, delivery particles subclasses thereof,and combinations thereof. For instance, in some embodiments, targetparticles are capture particles configured to bind a therapeutic moiety,a toxin, a desired cell type, or a synthetic particle. In someembodiments, target particles are carrier particles configured toculture stem cells. In some embodiments, target particles are configuredto deliver a therapeutic moiety.

Generally, target particles may be foreign particles introduced into thebiofluid to provide a therapeutic treatment. The therapeutic treatmentmay comprise delivering a therapeutic moiety, capturing a non-targetparticle, enriching the biofluid for a non-target particle, or culturinga cell type. The therapeutic moiety may comprise a drug, a chemicallyactive substance, or a biologically active substance. The targetparticles may be configured to capture non-target particles forseparation or enrichment in the biofluid. As previously described, thetarget particles may be cell culture carrier particles configured toculture stem cells.

Non-target particles may comprise any and all particles in the biofluidnot selected as the target particle. Generally, non-target particles maycomprise plasma, proteins, bacteria, toxins, viruses, cells, or otherbiochemical particles. Non-target particles may comprise cells selectedfrom the group consisting of erythrocytes, platelets, granulocytes,monocytes, macrophages, leukemic cells, and leukocytes. In someembodiments, the non-target particles are platelets and erythrocytes.

Target particles may be approximately the same size as certain cells. Inorder to separate target particles from cells in a biofluid, efficiencymay be greatly increased by including an additive to alter or regulateat least one parameter of the biofluid. For instance, the additive mayalter the aggregation potential of non-target particles and/or thedensity of the biofluid. According to certain embodiments, the additiveis introduced in sufficient volume to regulate the density of thebiofluid to be substantially similar to the density of the targetparticles.

According to certain embodiments, target particles are separated fromnon-target particles and removed to produce a target particle depletedfluid. The non-target particles may comprise live cells, frozen cells,preserved cells, or cells grown in a cell culture. The target particledepleted fluid may comprise a lower concentration of target particles,as compared to the biofluid suspension, the biofluid treated with targetparticles, or the pretreated biofluid.

Generally, a biofluid, for example whole blood, comprises a highconcentration of erythrocytes. To produce a target particle depletedfluid, it may be desirable to selectively separate erythrocytes from thetarget particles.

The method of separating target particles from non-target particles in abiofluid may further comprise providing a biofluid. In some embodiments,the biofluid may be obtained from a donor subject. The donor subject'sbiofluid may be subjected to down-stream processes directly, or may becollected and stored for later processing. As used herein, “directly”refers to processing of the biofluid without subjecting the biofluid toa long-term storage period. For instance, the biofluid may be processedimmediately in an in-line arrangement, within minutes, or within hours.The biofluid may be stored for one day or more.

In some embodiments, the biofluid is collected from a donor subjectthrough an intraluminal line. Accordingly, the method may furthercomprise obtaining the biofluid from a donor subject through anintraluminal line. As used herein, an “intraluminal” line refers to atransfusion line connectable to a lumen of a subject. More specifically,an intraluminal line may be connectable to a body cavity, tubularstructure, or organ in the body, such as a vein, an artery, the bladder,or intestine. For instance, a transfusion line may be connectable to thecirculatory or gastrointestinal system of the subject. The intraluminalline includes, for example, intravenous lines, central venous lines,intravascular lines, intratissue lines, catheters, and transfusionlines. The intraluminal line catheter may be, for example, a peripheralindwelling catheter, an intravenous catheter, or a central venouscatheter.

As used herein, the term “subject” is intended to include human andnon-human animals, for example, vertebrates, large animals, andprimates. In certain embodiments, the subject is a mammalian subject,and in particular embodiments, the subject is a human subject. Althoughapplications with humans are clearly foreseen, veterinary applications,for example, with non-human animals, are also envisaged herein. The term“non-human animals” of the invention includes all vertebrates, forexample, non-mammals (such as birds, for example, chickens; amphibians;reptiles) and mammals, such as non-human primates, domesticated, andagriculturally useful animals, for example, sheep, dog, cat, cow, pig,rat, among others.

In accordance with certain embodiments, the biofluid may be obtainedfrom a standard blood processing device. For instance, the biofluid maybe obtained from an apharesis machine. The biofluid may be directlyobtained from a standard blood processing device and further processedimmediately, for example in an in-line arrangement. In otherembodiments, the biofluid may be obtained from a standard bloodprocessing device and stored for one day or more before being introducedinto the microfluidic separation chamber.

In some embodiments, the method further comprises selecting the biofluidfrom blood buffy coat, leukapheresis product, peripheral blood, wholeblood, lymph fluid, synovial fluid, spinal fluid, bone marrow, ascitiesfluid, and combinations or subcomponents thereof. The biofluid maycomprise a synethetic medium comprising a cell suspension. For instance,the biofluid may comprise a cell culture medium. In some embodiments,the biofluid may comprise a subcomponent of a biofluid. For instance,the biofluid may comprise cell enriched biofluid, cell depletedbiofluid, diluted biofluid, concentrated biofluid, filtered biofluid,purified biofluid, or otherwise treated biofluid.

As used herein, leukapheresis product refers to a blood product whichhas undergone an apheresis separation process. The apheresis separationprocess may have been performed to deplete or enrich for leukocytes.Thus, the leukapheresis product may comprise leukocyte enrichedapheresis product or leukocyte depleted apheresis product. In someembodiments, the leukapheresis product may comprise synthetic biofluid.In some embodiments, the leukapheresis product may be purchased from amanufacturer. In some non-limiting embodiments, the leukapheresisproduct is LeukoPak™ leukapheresis product, as distributed by AllCells(Alameda, Calif.).

According to certain embodiments, the method may further compriseintroducing target particles into the biofluid. In some embodiments, thetarget particles may be introduced after the biofluid is collected andbefore further processing, according to the methods described herein.

The target particles may be introduced in-line, upstream from thepretreatment of the biofluid with an additive. The biofluid comprisingtarget particles may be processed or stored for a period of time beforeintroducing the additive into the biofluid.

In some embodiments, the biofluid may comprise target particles bound tonon-target particles. Such an embodiment is envisioned, for example,when the target particles comprise carrier, capture, delivery, orenrichment particles. The method may further comprise detaching thenon-target particles from the target particles before separation withinthe fluid. For instance, the method may comprise treating the biofluidto unbind the non-target particles for separation. The biofluid may betreated with a biochemical additive capable of unbinding the non-targetparticles. The biofluid non-target particles may be unbound from thetarget particles physically, for example by releasing a magnetic force.

The method of separating target particles from non-target particles in abiofluid may further comprise pretreating the biofluid. In someembodiments, pretreating the biofluid comprises introducing an additiveinto the biofluid to alter at least one of size of the target particles,size of the non-target particles, compressibility of the biofluid,compressibility of the target particles, compressibility of thenon-target particles, aggregation potential of the target particles, andaggregation potential of the non-target particles. The method mayfurther comprise introducing an additive into the biofluid to alter atleast one of density of the biofluid, density of the target particles,density of the non-target particles. The additive may be cell-friendly.For instance, in some embodiments, the concentration of additiveintroduced into the biofluid is generally safe for intraluminalinjection into a subject. In some embodiments, the additive selected isphysiologically acceptable and generally safe for intraluminal injectioninto a subject.

Generally, the method may comprise introducing an additive to modify thebiofluid or particle chemistry, to enhance separation of targetparticles from non-target particles. For instance, the biofluid'selectrolyte concentration (i.e. salinity or tonicity) may be adjusted,such that the particle or a desired cell type is enlarged, swollen,crenated, sphered, or rigidified in response. For instance, the changein one or more physical properties of the non-target particle cell typemay affect the response of the cell to the applied acoustic force withinthe microfluidic separation channel, enabling a differential separationbetween the target particle and other non-target particles, includingcell types within the biofluid. The method may comprise selecting theadditive from the group consisting of a cell aggregator, deionizedwater, a detergent, a surfactant, a solution to regulate salinity of thebiofluid, a solution to regulate tonicity of the biofluid, a solution toregulate viscosity of the biofluid, a solution to regulate osmolarity ofthe biofluid, a solution to regulate ion concentration of the biofluid,and combinations thereof.

The method may comprise introducing an additive to alter size or shapeof the target particles or non-target particles. As previouslymentioned, a target particle or non-target particle may become swollen,crenated, sphered, or rigidified in response to the introduction of anadditive in the biofluid. The change in size or shape may facilitatediscrimination between the particles in the separation process. Anadditive may also be introduced to activate a desired non-targetparticle cell type, whereby, for example, an activated cell may belarger than a target particle. Thus, natural morphological changes dueto biochemically induced activation or the natural cell cycle may beexploited to separate target particles from non-target particles.

The method may comprise introducing an additive to alter sodium or ionconcentration of the biofluid. For instance, a concentrated sodiumchloride solution may be introduced to crenate and/or shrinkerythrocytes and other non-target particles by osmosis. Without wishingto be bound by a particular theory, it is believed that hemoglobincontained within erythrocytes will effectuate an increase in densitysimultaneously with a decrease in volume of the cell. Thus, it may bepossible to selectively increase the density of erythrocytes bydecreasing their size, to promote an enhanced separation of targetparticles from non-target particles, including erythrocytes.

In some embodiments, the method comprises introducing an additive toalter compressibility of the biofluid, target particles, or non-targetparticles. For instance, detergents and/or surfactants may be added toalter cell membrane mechanics, such that desired non-target particlecell types undergo a change in compressibility. In some embodiments,detergents or surfactants alter the cell membrane, such that desirednon-target particle cell types are more susceptible to changes in ionconcentration in the biofluid.

An additive may be introduced into the biofluid to alter aggregationpotential of the target particles or the non-target particles. As usedherein, “aggregation potential” refers to the mechanism by which adesired cell type or particle aggregates, agglutinates, adheres, orforms a complex with like particles. In some embodiments, theaggregation potential refers to a desired cell type's ability toaggregate with cells of the same or a different cell type. For instance,an additive may be introduced to alter or regulate the aggregationpotential of erythrocytes or platelets. Generally, many biofluidscomprise a high concentration of erythrocytes and/or platelets. Byaggregating the erythrocytes and/or platelets, a more efficientseparation from target particles may be achieved.

In some embodiments, the aggregation potential is altered or regulatedby an additive that prohibits a target particle, non-target particle, ora desired cell type from binding, aggregating, agglutinating, adhering,or forming a complex with a like or different particle. For instance,the aggregation potential may be altered or reduced by ananti-coagulant. In other embodiments, the aggregation potential may bealtered, enhanced, or regulated by a cell aggregator. As used herein, a“cell aggregator” refers to an additive that may bind, aggregate,adhere, agglutinate or form a complex with a desired cell type. A “cellaggregator” may also refer to an additive that may cause a desired celltype to bind, aggregate, adhere, agglutinate, or form a complex withlike or different cell types. The cell aggregator may cause cells toaggregate by activating natural biochemical pathways, by altering cellmechanics, or by reducing or screening electrostatic barriers betweencells in the pretreated biofluid.

In some embodiments, the method further comprises selecting the cellaggregator to be a long-chain polysaccharide. Long-chain polysaccharidesinclude, but are not limited to, dextran, polysucrose, hetastarch(hydroxyethyl starch), and Ficoll™ media, distributed by GE Healthcare(Chicago, Ill.). The long-chain polysaccharide may have a molecularweight between about 100 kD and about 500 kD. In some embodiments, thelong-chain polysaccharide has a molecular weight between about 250kD andabout 500 kD, between about 200 kD and about 400 kD, between about 300kD and about 400 kD. The long-chain polysaccharide may have a molecularweight of about 100 kD, about 200 kD, about 250 kD, about 300 kD, about400 kD, and about 500 kD. In some embodiments, the cell aggregatorcomprises a long-chain polysaccharide present at a concentration ofbetween about 0.5% (w/v) and about 25% (w/v). In some embodiments, thecell aggregator comprises a long-chain polysaccharide present at aconcentration of between about 1.0% (w/v) and about 20% (w/v), betweenabout 5.0% (w/v) and about 15% (w/v), between about 8.0% (w/v) and about12% (w/v). For instance, the cell aggregator may comprise a long-chainpolysaccharide present at about 0.5% (w/v), about 1.0% (w/v), about 2.0%(w/v), about 5.0% (w/v), about 8.0% (w/v), about 10% (w/v), about 12%(w/v), about 15% (w/v), about 20% (w/v), about 24% (w/v), and about 25%(w/v).

In some embodiments, the method further comprises selecting the cellaggregator to be a platelet aggregator or a cell adhesion molecule(CAM). The CAM may be released or obtainable from an activated plateletgranule. Such CAMs aggregate platelets by known natural mechanisms.Platelet activation may induce the platelet to releases granules andexposed the contents of platelet granules on the outside of the cell.CAMs may then promote platelet aggregation through platelet-fibrin andplatelet-platelet binding. CAMs may be released from an activatedplatelet granule by biochemically inducing their release, for examplethrough activation by addition of thrombin, Type II collagen oradenosine diphosphate, or by introducing natural or synthetic CAMsobtained from a distributor into the biofluid. The CAMs released orobtainable from an activated platelet granule may include, but are notlimited to, P-selectin and von Willebrand factor. Platelet activatorsinclude, but are not limited to, adenosine diphosphate, thrombin, TypeII collagen, and ristocetin.

An additive may be introduced into the biofluid to alter density of thebiofluid. In some embodiments, the additive is selected from a densitygradient medium, a density additive, and combinations thereof. Densitygradient media is a media for cell isolation, generally used in thepractice of centrifugal separation. Density gradient media are wellknown in the art and include, for example, ACCUSPIN™ media, Histodenz™media, OptiPrep™ media, and Histopaque® media distributed bySigma-Aldrich (St. Louis, Mo.), Ficoll-Paque™ media and Percoll™ mediadistributed by GE Healthcare (Chicago, Ill.), RosetteSep™ media andLymphoprep™ media distributed by STEMCELL Technologies (Vancouver,Canada). The list of density gradient media is merely exemplary andnon-exhaustive. A density additive may comprise a reagent having adifferent density than the biofluid, or configured to regulate or alterthe density of the biofluid. For instance, the density additive maycomprise pure water, deionized water, a salt, a saline buffer solution,or a nonionic iodinated compound. Nonionic iodinated compounds include,but are not limited to, diatrizoic acid, meglumine diatrizoate, andiodixanol. According to certain embodiments, the density additive isselected to be cell-friendly, such that it does not increase osmolarityof the biofluid to a degree that would be harmful to the cells.

In some embodiments, the additive is introduced to alter or regulate thedensity of the biofluid to be within a range of the density of thetarget particles or non-target particles. The additive may be introducedto regulate the density of the biofluid to substantially match a densityof the target or non-target particles. For example, the density may beregulated such that target particles approach neutral buoyancy in thebiofluid, reducing the acoustic force acting on them, as compared to theforce acting on the non-target particles. The density of the biofluidmay be regulated to a density of between about 1.00 g/mL and about 1.15g/mL. In some embodiments, the density of the biofluid is regulated to adensity of between about 1.00 g/mL and about 1.10 g/mL, between about1.10 g/mL and about 1.15 g/mL, between about 1.02 g/mL and about 1.09g/mL, between about 1.03 g/mL and about 1.08 g/mL, between about 1.04g/mL and about 1.07 g/mL, and between about 1.045 g/mL and about 1.065g/mL. Specifically, the density of the biofluid may be regulated oraltered to a density of about 1.00 g/mL, about 1.01 g/mL, about 1.02g/mL, about 1.03 g/mL, about 1.04 g/mL, about 1.05 g/mL, about 1.06g/mL, about 1.07 g/mL, about 1.08 g/mL, about 1.09 g/mL, about 1.10g/mL, about 1.12 g/mL, and about 1.15 g/mL.

Pretreating the biofluid may further comprise introducing an additive toalter density of the target particles or non-target particles. Theadditive may be introduced to alter or regulate the density of biofluidand particles to be within a range of each other, for instance to makethe particles approach neutral buoyancy within the fluid. A diluent,salt, or saline solution may be introduced to alter or regulate thedensity of target particles or non-target particles to illicit a certainresponse from a desired non-target particle cell type or to have adensity within a range of the density of the biofluid. For instance,sodium or an ion concentration may be reduced, for example by dilutionwith deionized water, to swell a first type of particle by osmosis whileother particles may use known natural mechanisms to regulate their size,increasing size discrimination between the two particle types. Inanother non-limiting example, erythrocytes swell more readily thantarget particles, and the additive may facilitate removal of theleukemic cells.

The method may comprise introducing an additive to alter both density ofthe biofluid and aggregation potential of the non-target particles. Insome embodiments, the combination of a density additive and a cellaggregator produces a synergistic effect, whereby the method produces amore efficient separation of target particles from non-target particlesand a higher concentration of target particles in the target particleenriched fluid than would be expected from the combination of botheffects. For instance, in a method of separating target particles fromleukocytes and erythrocytes, an additive or a combination of additivesmay be introduced to alter the density of the biofluid and to aggregateerythrocytes. The density additive may enhance the separation of thetarget particle, from the non-target particles (for example, theerythrocytes and leukocytes), while the cell aggregator may effectivelyincrease the acoustic scattering radius of the non-target particles toenhanced separation of the non-target particles over the targetparticles. The individual additives, when used separately, may notprovide sufficient separation of target particles from non-targetparticles including erythrocytes and leukocytes, but the combination maypromote an enhanced effective differential separation of targetparticles from non-target particles.

In some embodiments the additive may further comprise affinity basedcapture particles. Generally, the affinity based particles are safe forintraluminal injection into a subject. For instance, the additive maycomprise biochemical moieties, such as antibodies, that bind targetparticles or non-target particles. The cell aggregator may comprise asolution comprising antibodies that bind and aggregate target particlesor non-target particles. In some embodiments, the antibodies bind andaggregate a desired target particle. The additive may comprise emulsiondroplets, gel particles, or lipid encapsulated oil vesicles. In someembodiments, the affinity based capture particle is safe forintraluminal injection. The affinity based capture particle may beengineered to be “anti-focusing” or “positively focusing” by designingit with low density or high density. The low density “anti-focusing”capture particle may experience acoustophoretic forces in the oppositedirection as the target particles or non-target particles. The highdensity “anti-focusing” capture particle may experience migration to thepressure anti-node, while target particles or non-target particlesmigrate toward the pressure node. In some embodiments, an acousticanalog to magnetic separation may comprise “positively focusing” captureparticles. For instance, a “positively focusing” capture particle may beused to trap a desired target particle, such that selected non-targetparticles remain held in the separation channel, while other targetparticles flow through. The held non-target particles may be released ata later time. In some embodiments, a large capture particle molecule maybind to many points on the surface of a target particle, and may alterthe acoustophoretic force exhibited on the particle by changing itseffective diameter.

The method of separating target particles from non-target particles mayfurther comprise flowing biofluid into an inlet of a microfluidicseparation channel. For instance, the method may comprise flowing thepretreated biofluid into the microfluidic separation channel. Thebiofluid may have a flow rate of between about 0.03 mL/min to about 0.5mL/min. In some embodiments, the biofluid may have a flow rate throughthe microfluidic separation channel of between about 0.05 mL/min toabout 0.4 mL/min, about 0.1 mL/min to about 0.3 mL/min. The biofluid mayhave a flow rate through the microfluidic separation channel of about0.03 mL/min, 0.05 mL/min, 0.08 mL/min, 0.1 mL/min, 0.2 mL/min, 0.3mL/min, 0.4 mL/min, 0.5mL/min, or any range therebetween.

The method may further comprise applying acoustic energy to themicrofluidic separation channel. In some embodiments, the acousticenergy is applied in the form of an acoustic wave.

The acoustic wave may be applied at an angle relative to the flow offluid through the separation channel. The angle and magnitude of theacoustic wave may be engineered based on size of the device, size of thechannel, or flow rate of fluid through the channel. In some embodiments,the acoustic energy may be applied in a direction substantiallytransverse to the biofluid flow through the microfluidic separationchannel. The acoustic wave may be a standing acoustic wave. In someembodiments, the acoustic energy may be applied to the microfluidicseparation channel continuously. The continuous application of acousticenergy may allow for a greater efficiency of separation. In alternateembodiments, the acoustic energy may be applied to the microfluidicseparation channel intermittently or on a timed schedule. Theintermittent energy application may allow for particles to move freelythrough the channel if there is a blockage.

The applied acoustic energy may act on the cells and particles withinthe biofluid to drive them according to size, density, and/orcompressibility. In some embodiments, the method may compriseaccumulating target particles within a primary stream along theseparation channel. In some embodiments, the method may compriseaccumulating non-target particles within a secondary stream along theseparation channel. The accumulation of a target or non-target particlewithin a desired stream along the separation channel may be engineeredby adjusting parameters such as wavelength, frequency, amplitude, powerlevel, or other modulation of the applied acoustic energy.

Depending on the target particles or non-target particles selectedaccording to the method, one class of particles may accumulate inresponse to a pressure node or anti-node generated by the acousticenergy. For instance, target particles may accumulate within a primarystream in response to a pressure node, and non-target particles mayaccumulate within a secondary stream in response to a pressureanti-node. Generally, particles, including cells, will be driven by theacoustic energy in response to their contrast factor. Particles maymigrate at a rate which is proportional to the magnitude and sign oftheir contrast factors. In some embodiments, particles with a positivecontrast factor are driven to pressure nodes, while particles with anegative contrast factor are driven to pressure anti-nodes. Particleswith a greater magnitude contrast factor are generally driven at afaster rate than particles with a lesser magnitude contrast factor.

The rate at which particles are driven in response to their acousticenergy generally depends on particle size, density, and compressibility.Briefly, the contrast factor is based on the bulk modulus (K) anddensity (ρ) of a particle. When suspended in a fluid, the contrastfactor (φ) for the particles is calculated with the below equation:

$\phi = {\frac{{5\rho} - {2 \cdot 1.02}}{{2\rho} + 1.02} + \frac{2.2}{K}}$

In some embodiments, the method of separating target particles fromnon-target particles in a biofluid comprises collecting the at least oneprimary stream comprising the target particles. Generally, the biofluidentering the microfluidic separation channel is a well-mixed primarystream, comprising desegregated target particles and non-targetparticles. Upon experiencing acoustic energy, target particles andnon-target particles may generally accumulate into fractions of thegeneral stream of biofluid. The fraction or fractions of biofluidflowing through the microfluidic separation channel selectively enrichedin target particles are defined as the primary stream. There may be morethan one fraction of biofluid within the microfluidic separation channelenriched in target particles. For instance, target particles may bedriven to a pressure node at the center of the channel in oneembodiment, and target particles may be driven to the pressureanti-nodes at the periphery of the channel in an alternate embodiment.The location of pressure nodes and anti-nodes within the channel may bedesigned by positioning the acoustic energy or by selecting frequencyand wavelength of the acoustic waves. The primary stream comprisingtarget particles may be collected for storage, for research, forrecycling, or as waste

Similarly, in some embodiments, the method of separating targetparticles from non-target particles comprises collecting the at leastone secondary stream comprising non-target particles. The fraction orfractions within the biofluid selectively depleted in target particles,and selectively enriched in non-target particles are defined as thesecondary stream. In certain embodiments, the target particles andnon-target particles have opposing contrast factors. With opposingcontrast factors, the target particles and non-target particles may bedriven in opposite directions, or one may be driven away from thegeneral stream, for example to the center or the periphery of thechannel. In other embodiments, the target particles and non-targetparticles have contrast factors of a different magnitude, but the samesign. In these embodiments, one class of cells may be driven away at afaster rate than the other, defining the primary and secondary streams.The secondary stream may be collected for storage, immediate use, fortransfusion into a subject, or for further research The method maycomprise collecting the primary stream comprising target particles andfurther comprise separately collecting the at least one secondary streamcomprising the non-target particles.

In some embodiments, the target particle enriched primary stream iscollected for recycling target particles. Where the target particles arebound to other particles, the particles may be detached, as previouslydescribed, before recycling. The recycled target particles may beintroduced into the biofluid, upstream from pretreating with anadditive, to provide a therapeutic treatment to at least one componentof the biofluid.

According to certain embodiments, target particle depleted fluid may bepost-treated and delivered to a recipient subject. For instance, thesecondary stream may be post-treated and delivered to a recipientsubject. Post-treating a fluid may comprise a process such as washing,separating, concentrating, diluting, heating, purifying, or filteringcapable of removing toxins, contaminants, or harmful chemical compoundsfrom the fluid. In general, a fluid is post-treated to produce aphysiologically acceptable fluid that may be directly delivered to arecipient subject, for example via an intraluminal line as previouslydescribed. The post-treated fluid may be stored for delivery to arecipient subject at a later time.

In some embodiments, the target particle depleted fluid is post-treatedto produce a therapeutic fluid. Post-treating the fluid may compriseviral transduction, gene transfer, or gene editing of the targetparticles to produce a therapeutic, physiologically acceptable fluid fordelivery to a recipient subject, as previously described.

In some embodiments, the recipient subject is the same as the donorsubject. In other embodiments, the donor subject and the recipientsubject are not the same. The donor subject and the recipient subjectmay generally be physiologically compatible.

The method may be performed in line such that the biofluid is collectedfrom a subject, target particles are introduced into the biofluid toprovide a therapeutic treatment, and the biofluid comprising targetparticles is directly pretreated, target particles are separated fromnon-target particles in the biofluid by a method as described herein toproduce a target particle depleted fluid, the fluid depleted of targetparticles may be post-treated, and the post-treated fluid may bedirectly delivered back to the subject. In some embodiments, the methodis performed essentially as previously discussed, however the targetparticles are separated from non-target particles to produce a targetparticle enriched fluid, which may be post-treated and delivered back tothe subject.

According to certain embodiments, the method further comprises flowing asecond fluid adjacent to the biofluid into an inlet of the microfluidicseparation channel. The inlet may be an inlet separate from the biofluidinlet of the microfluidic separation channel. The biofluid and thesecond fluid may flow through the separation channel in substantiallyparallel form. For instance, both fluids may flow through the separationchannel at opposite peripheries of the channel, the second fluid mayflow through both peripheries of the channel, or the second fluid mayflow in the center of the channel. The biofluid and the second fluid mayflow through the separation channel in substantially laminar form. Asused herein, substantially laminar flow includes substantially orderedflow. Laminar flow may have a Reynolds number (Re) less than about 2100.In certain embodiments, laminar flow has a Reynolds number (Re) lessthan about 4000.

In certain embodiments, the second fluid is an inert fluid that maycomprise water, deionized water, or phosphate buffered saline (PBS). Thesecond fluid may have its density adjusted with a density gradientmedium or density additive, independently from the pre-treated biofluid.The applied acoustic energy may drive target or non-target particlesfrom the biofluid into the essentially parallel flowing second fluidinitially comprising no cells, such that the second fluid, nowcomprising selectively separated cells, may exit the microfluidicseparation channel through a separate outlet. Where the target particlesare driven into the second fluid, the second fluid comprising targetparticles is essentially the primary stream. Conversely, where thenon-target particles are driven into the second fluid, the second fluidis essentially the secondary stream.

According to certain embodiments, the methods described herein may beperformed in a staged separation or in series. Specifically a targetparticle enriched fluid or a target particle depleted fluid may befurther processed by pretreating with an additive, flowing through asecond microfluidic separation channel, and applying acoustic energy.The additive introduced into the fluid in the downstream operation maybe the same or a different additive as the one introduced into thebiofluid in the first pass separation process. Additionally, the targetparticles selected in the first pass process may be the same ordifferent as those selected in the second pass process. As anon-limiting example, a biofluid may be pretreated and flowed through amicrofluidic separation channel to produce a target particle depletedfluid. The output target particle depleted fluid may further be flowedthrough a second microfluidic separation channel to remove a differenttarget particle. As another non-limiting example a biofluid may beflowed through a microfluidic separation channel to produce a selectedcell type enriched fluid. The cell enriched fluid may be flowed througha second microfluidic separation channel to remove target particles andproduce a further cell enriched fluid.

In some embodiments, the first pass target particle enriched or targetparticle depleted fluid is recycled and reintroduced into the biofluidor into the pretreated biofluid to flow through the microfluidicseparation channel as a blend. For instance, the target particledepleted fluid may be recycled and reintroduced into the biofluid orpretreated biofluid to flow through the microfluidic separation channela second time.

According to certain embodiments, the method further comprises dosingthe at least one secondary stream comprising cells with a reagent toproduce a dosed suspension. The at least one secondary stream may be atarget particle depleted fluid. The reagent may be selected from anantigen or activation reagent configured to biochemically induce cellactivation. The biochemically induced activation may allow for selectionof subclasses of types of cells in a second pass separation, forinstance lymphocytes or T cells, by exploiting the morphological changesof activated cells. In some instances, activated cells may be largerthan non-activated cells and cell size may vary throughout the cellcycle. The difference in size may allow for differential separation ofcells with acoustic energy.

The method may further comprise flowing the target particle depletedfluid comprising cells through a second microfluidic separation channelor through microfluidic separation channels arranged in series andapplying acoustic energy to each separation channel. The dosedsuspension may allow for selection of target particles at a certainstage of the cell cycle.

For instance, in some embodiments of the method, the target particledepleted fluid may comprise lymphocytes and the method may furthercomprise separating activated lymphocytes from non-activated lymphocytesin the secondary stream. The method may further comprise dosing thelymphocyte enriched fluid with a reagent to produce the dosedsuspension, flowing the dosed suspension into an inlet of a secondmicrofluidic separation channel, and applying acoustic energy to thesecond microfluidic separation channel. Activated lymphocytes mayaccumulate within at least one primary stream along the secondseparation channel and non-activated lymphocytes may accumulate withinat least one secondary stream along the second separation channel.

In accordance with another aspect, there is provided a system formicrofluidic particle separation. The system may be configured toseparate target particles from non-target particles in a biofluid. Insome embodiments, the system comprises at least one microfluidicseparation channel comprising at least one inlet and at least oneoutlet. The at least one outlet may be a branched outlet, branching in adirection substantially away from the separation channel. In someembodiments, the microfluidic separation channel comprises a firstoutlet and a second outlet. The at least one inlet may be configured toreceive biofluid and the at least one outlet may be configured todischarge the biofluid that has been subjected to acoustic energy. Asthe fluid flows through the microfluidic separation channel, it may besubjected to acoustic energy that drives the target particles and/ornon-target particles towards pressure nodes and anti-nodes within thechannel. In some embodiments, the first outlet is configured todischarge target particle enriched fluid and the second outlet isconfigured to discharge target particle depleted fluid.

The microfluidic separation channel may be formed of rigid materials.The rigid materials may have a high acoustic contrast with the biofluid.In alternate embodiments, the microfluidic separation channel may beformed of relatively elastic materials. The more elastic materials mayhave a lower acoustic contrast with the biofluid, however they may formgood acoustic resonators that provide low acoustic impedance and providerelatively little wave energy loss in wave transfer. The materials toform the microfluidic separation channel may include silicon, glass,metals, thermoplastics, and combinations thereof. In some embodiments,the microfluidic separation channel may be formed of a thermoplasticmaterial. The thermoplastic microfluidic separation channel may besmall, disposable, relatively safer to handle than, for example, theglass or metal separation channels, and relatively less expensive tomanufacture than the silicon, glass, or metal separation channels. Insome embodiments, the thermoplastic microfluidic separation channels aremanufactured by injection molding. The thermoplastic material maycomprise polystyrene, acrylic (polymethyl methacrylate), polysulfone,polycarbonate, polyethylene, polypropylene, cyclic olefin copolymer,silicone, liquid crystal polymer, polyvinylidene fluoride, andcombinations thereof. The microfluidic separation channel may bedisposable.

In some embodiments, the microfluidic separation channel has a channelwidth of between about 0.2 mm to about 0.8 mm. The microfluidicseparation channel may be about 0.2 mm, about 0.3 mm, about 0.4 mm,about 0.5 mm, about 0.6 mm, about 0.7 mm, or about 0.8 mm wide. In someembodiments, the microfluidic separation channel is between about 15 mmand about 35 mm long. The microfluidic separation channel may be about15 mm, about 20 mm, about 25 mm, about 30 mm, or about 35 mm long. Themicrofluidic separation channel width may be correlated to the acousticwave wavelength, such that each channel contains a pressure-node and/orpressure anti-node generated by the acoustic energy. The operatingfrequency may be chosen so that the acoustic wavelength in the fluid isabout twice the width of the microchannel. In an exemplary embodimentthe operating frequency is chosen so that the acoustic wavelength in thefluid is about 3-4 times the width of the microchannel.

The system may further comprise a source of biofluid in fluidcommunication with the microfluidic separation channel. The source ofthe biofluid may be a vessel or chamber in fluid communication with theat least one inlet of the microfluidic separation channel, configured todeliver biofluid to the separation channel. The source of the biofluidmay be a mixing chamber configured to receive an additive or a secondfluid to be introduced into the biofluid prior to flowing the biofluidthrough the microfluidic separation channel. The source of the biofluidmay be heated, cooled, or mixed.

In some embodiments, the source of the biofluid is fluidly connecteddownstream of an intraluminal line, and configured to receive biofluiddirectly from a donor subject. The source of the biofluid may further befluidly connected downstream to a biofluid sample, for instance a samplecollected in a bag, vessel, tank, or other chamber.

The system may further comprise a source of a target particle. Thesource of the target particle may be configured to deliver targetparticles to the biofluid for therapeutic treatment to the at least onecomponent of the biofluid. The source of a target particle may comprisea target particles suspended in a medium. The medium may betherapeutically active or may be inert. Generally, the medium may bephysiologically acceptable for intraluminal injection.

In some embodiments, the system further comprises a target particleprocessing chamber. The target particle processing chamber may beconfigured to unbind non-target particles from target particles prior toseparation. In some embodiments, the target particle processing chamberis fluidly connected to a source of a treatment fluid configured tofacilitate detachment of the non-target particles from the targetparticles. For instance, the treatment fluid may comprise a biochemicaladditive, capable of detaching non-target particles from targetparticles. The biochemical additive may comprise a chemically activemoiety or a biologically active moiety. The processing chamber mayfurther be equipped with materials to physically detach non-targetparticles from target particles, for example by releasing a magneticforce.

In some embodiments, the system comprises a source of additive in fluidcommunication with the source of the biofluid, configured to introduceat least one additive into the biofluid.

The additive contained in the source of the biofluid may be an additivecapable of altering or regulating at least one of size of the targetparticles, size of the non-target particles, compressibility of thebiofluid, compressibility of the target particles, compressibility ofthe non-target particles, aggregation potential of the target particles,and aggregation potential of the non-target particles, as previouslydiscussed. The additive may further be capable of altering or regulatingat least one of density of the biofluid, density of the targetparticles, density of the non-target particles. The source of theadditive may be a chamber, vessel, or tank comprising the additive. Insome embodiments, the system comprises more than one source of anadditive, each source configured to introduce a separate additive intothe biofluid. In some embodiments, the source of the additive may beheated, cooled, or mixed.

The system may further comprise at least one acoustic transducer coupledto a wall of the microfluidic separation channel. The acoustictransducer may be positioned to apply a standing acoustic wavetransverse to the microfluidic separation channel. In some embodiments,the acoustic transducer is capable of emitting acoustic energy thatdrives cells and/or particles to a pressure node or anti-node. Theacoustic transducer may comprise a piezoelectric vibrating elementconfigured to emit acoustic energy. The denser and larger particles andcells may migrate towards the center of the separation channel inresponse to the acoustic energy emitted by the piezoelectric transducer.In some embodiments, the acoustic transducer is configured to emitacoustic energy between about 0.2 MHz and about 4.0 MHz. For instance,the acoustic transducer may emit acoustic energy between about 0.5 MHzand about 3.0 MHz or between about 0.5 MHz and about 1.5 MHz. Theacoustic transducer may be configured to provide standing acoustic waveshaving a wavelength that is twice as long as the microfluidic separationchannel width.

The microfluidic separation channel may further comprise one or moreheat sinks configured to dissipate heat generated by the acoustictransducer. The heat sink may be configured to dissipate enough heatfrom the acoustic transducer to prevent the transducer from warmingfluids flowing through the separation channel. In some embodiments, theheat sinks comprise thermoelectric coolers. In some embodiments, thesystem includes fluidic lines that flow into the heat sink to providefluidic cooling to the heat sink.

Systems that comprise more than one microfluidic separation channel maycomprise one acoustic transducer coupled to each microfluidic separationchannel or one or more acoustic transducers coupled to a collection ofmicrofluidic separation channels.

In some embodiments, the system comprises at least two microfluidicseparation channels. The at least two microfluidic separation channelsmay be arranged in a parallel arrangement downstream of the source ofthe biofluid. In such embodiments, the system may further comprise amanifold configured to distribute biofluid to the at least twomicrofluidic separation channels. The manifold may be configured toreceive a biofluid or pretreated biofluid sample and evenly distributethe sample to downstream microfluidic separation channels. In someembodiments, the manifold may be configured to continuously receive anddistribute fluid, and in other embodiments the manifold may beconfigured to receive and distribute fluid in batches. The manifoldconfigured to receive and distribute fluid in batches may be on aregular timer or may distribute fluid batches as it receives sufficientfluid.

In some embodiments, the manifold is configured to distribute thebiofluid in response to the input biofluid load on the system. In someembodiments, the input biofluid load comprises between about 1 mL toabout 10L of fluid. In some embodiments, the input biofluid load on thesystem may have a flow rate of between about 0.1 mL/min to about 200mL/min. Each microfluidic separation channel may be configured toreceive flow rates of between about 0.1 mL/min to about 0.5 mL/min. Thesystem may further comprise at least one sensor configured to measure aninput biofluid load on the system. The input biofluid load sensor may bein electrical communication with the manifold, such that the manifoldmay distribute the biofluid to the two or more microfluidic separationchannels in response to the measurement of the input biofluid loadreceived from the input biofluid load sensor.

In some embodiments, the system further comprises at least one sensorconfigured to measure at least one parameter of the input biofluid. Forinstance, the biofluid sensor may be configured to measure at least oneof density of the biofluid, HCT % of the biofluid, concentration oftarget particles, or concentration of non-target particles in thebiofluid. In some embodiments, the biofluid sensor is configured tomeasure optical transmission or absorption of the biofluid at apredetermined optical wavelength. The at least one biofluid sensor maybe positioned at the system input and configured to measure parametersfrom the input biofluid load, or may be positioned within the source ofthe biofluid and configured to measure parameters from the biofluid orpretreated biofluid. The system may further comprise a control module inelectrical communication with the biofluid sensor. The control modulemay further be in electrical communication with the source of additive,and configured to introduce a predetermined volume of the additive intothe biofluid in response to the measurement of the at least oneparameter of the input biofluid.

In certain embodiments the additive is capable of altering or regulatingat least one of density of the biofluid, density of the targetparticles, density of the non-target particles, and the predeterminedvolume of the additive is determined to alter or regulate the biofluidto have a desired density or concentration of target particles ornon-target particles. For instance, the predetermined volume of theadditive may be determined to allow target particles or non-targetparticles to approach neutral buoyancy in the biofluid. Thepredetermined volume of the additive may be determined to regulate thedensity of the biofluid to substantially match the density of the targetparticles or non-target particles. In some embodiments, thepredetermined volume of the additive is determined to alter or regulatethe density of the biofluid to a density of between about 1.00 g/mL andabout 1.15 g/mL or to density ranges or values within this range, aspreviously discussed.

In some embodiments, the additive is capable of altering or regulatingat least one of HCT % of the biofluid, concentration of the targetparticles, or concentration of the non-target particles, and thepredetermined volume of the additive is determined to alter or regulatethe HCT % of the biofluid to be less than about 10%. For instance, thepredetermined volume of the additive may be determined to alter orregulate the HCT % of the biofluid to be less than about 30%, less thanabout 25%, less than about 20%, less than about 15%, less than about10%, or less than about 5%.

According to certain embodiments, the system further comprises at leastone sensor configured to measure a parameter of an output suspension.The output suspension may be target particle enriched fluid or targetparticle depleted fluid exiting the microfluidic separation channelthrough the at least one outlet, or product or waste exiting the system.The sensors may measure at least one of HCT %, concentration of targetparticles, or concentration of non-target particles in the outputsuspension. In some embodiments, the sensors may measure at least one ofdensity of the output suspension, density of the target particles,density of the non-target particles, size of the target particles, sizeof the non-target particles, compressibility of the output suspension,compressibility of the target particles, compressibility of thenon-target particles, and concentration of the additive in the outputsuspension. In some embodiments, the sensors may measure opticaltransmission or absorption of the output suspension at a predeterminedwavelength.

The system may further comprise a control module in electricalcommunication with the output suspension sensor. The control module maybe in electrical communication with the acoustic transducer, andconfigured to alter or regulate at least one input parameter of theacoustic transducer. For instance, the control module may alter orregulate the power, voltage, or frequency delivered to the acoustictransducer in response to a measurement of a parameter of the outputsuspension. The control module may further shut on or off the acoustictransducer in response to a measurement of a parameter of the outputsuspension. For instance, the control module may act in response to ameasurement of HCT %, concentration of target particles, orconcentration of non-target particles in the output suspension. Thecontrol module in communication with the output suspension sensor may bethe same or different from the control module in communication with thebiofluid sensor. In some embodiments, any control module may be designedto act in response to a measurement from any sensor within the system.For instance, the control module configured to introduce a predeterminedvolume of additive into the biofluid may further be in electricalcommunication with the output suspension sensor or input biofluid loadsensor, and configured to act in response to a measurement receivedtherefrom. In another embodiment, the control module configured to be inelectrical communication with the acoustic transducer may also be inelectrical communication with other sensors and configured to act inresponse to a measurement received from the biofluid load sensor or thebiofluid sensor.

In some embodiments, the predetermined volume of the additive or thepower, voltage or frequency delivered to the acoustic transducer arecontrolled to regulate the HCT % of the output suspension. For instance,the system may be controlled to provide an output suspension having adesired HCT % of less than about 20%, less than about 10%, or less thanabout 1%. In some embodiments, the HCT % of the output suspension iscontrolled to be less than about 10%, less than about 9%, less thanabout 8%, less than about 7%, less than about 6%, less than about 5%,less than about 4%, less than about 3%, less than about 2%, or less thanabout 1%. The desired output suspension HCT % will depend on the exactbiofluid flowed through the system and the input biofluid HCT %. Forexample, if the input biofluid is whole blood having a HCT % of 45%, thesystem may be controlled to provide an output suspension having a HCT %of about 5%.

The system may further comprise a source of a second fluid in fluidcommunication with the at least one inlet of the at least onemicrofluidic separation channel. The source of the second fluid may be avessel, tank, or chamber in fluid communication with the microfluidicseparation channel, the source of the biofluid, or a line connecting thesource of the biofluid with the at least one inlet of the microfluidicseparation channel. The source of the second fluid may be configured tointroduce the second fluid into the biofluid. In some embodiments, thebiofluid and the second fluid flow in substantially parallel,substantially laminar flow, as previously discussed. The second fluidmay be any fluid, as previously discussed.

In some embodiments, the system may further comprise a first and secondcollection channel in fluid communication with the at least one outletof the microfluidic separation channel. The collection channel may be afluid line configured to deliver output suspension to a vessel, recycleline, or fluidly connectable with an intraluminal line configured todeliver output suspension to a subject. A collection vessel may be influid communication with the first or second collection channel. Thecollection vessel may be used to store, freeze, heat, or otherwise keepoutput suspension.

According to certain embodiments, the system further comprises a recycleline. In some embodiments, the recycle line is a line or channelconfigured to deliver output suspension back to the source of thebiofluid for a second pass separation. The recycle line may beconfigured to deliver output suspension back to the at least one inletof the microfluidic separation channel. The output suspension that isrecycled may be target particle enriched fluid or target particledepleted fluid.

The system may further comprise a recycle line configured to delivertarget particle enriched fluid back to the biofluid to provide atherapeutic treatment to at least one component of the biofluid. Therecycle line may be equipped with a target particle processing chamberto detach any non-target particles from the target particles, beforedelivering the target particles back to the biofluid. In someembodiments, the recycle line is equipped with a parallel system formicrofluidic particle separation, as described herein.

In some embodiments, the system comprises a post-treatment chamber. Thepost-treatment chamber may be configured to post-treat output suspensionto produce a post-treated fluid, physiologically acceptable fluid, ortherapeutic fluid, as previously described.

The system may comprise one or more pumps to direct the biofluid throughthe system. The one or more pumps may be an infusion pump configured togenerate sufficient pressure to force the biofluid through the system.In some embodiments, the pump generates sufficient pressure to introducethe output suspension into the recipient subject through theintraluminal line.

The system may be connectable to more than one intraluminal line toproduce an in-line system for separation of particles. For instance, thesystem may be connectable to an intraluminal line configured to extractbiofluid from a donor subject and deliver it to the source of thebiofluid for processing. The system may be connectable to anintraluminal line configured to deliver an output suspension, forexample target particle enriched fluid or target particle depletedfluid, to the recipient subject. In some embodiments, the recipientsubject may be the same as the donor subject, and the biofluidprocessing is performed in line and in real time.

In some embodiments, the system comprises more than one microfluidicseparation channel arranged in series. The more than one microfluidicchannel in series may be configured to separate target particles fromnon-target particles in consecutive separation channels to produce afluid with a higher degree of target particle depletion. In someembodiments, the more than one microfluidic separation channel in seriesis configured to deliver target particle depleted fluid to downstreammicrofluidic separation channels. In alternate embodiments, the morethan one microfluidic separation channel in series is configured todeliver target particle enriched fluid to downstream microfluidicseparation channels. In some embodiments, the microfluidic separationchannels in series are stacked to process relatively larger volumes ofbiofluid. The stacked configuration allows branched outlets of theseparation channel to be easily connectable to branched inlets of adownstream separation channel.

In accordance with another aspect, there is provided a kit forseparation of target particles from non-target particles. The kit maycomprise at least one microfluidic separation channel connected to anacoustic transducer, a source of an additive fluidly connectable to theat least one inlet of the microfluidic separation channel, andinstructions for use. The at least one microfluidic separation channelmay be configured to separate target particles from non-targetparticles, as previously described herein. The source of the additivemay be a vessel, chamber, or channel, as previously discussed herein andmay comprise at least one additive, as previously discussed herein. Thekit may further comprise any component of the system described herein,connectable to the microfluidic separation channel. For instance,according to certain embodiments, the kit may further comprise acollection channel, a collection vessel, a manifold system, a sensor, acontrol module, an intraluminal line, a pump, a source of a targetparticle, a target particle processing chamber, a post-treatmentchamber, or fluid lines to fluidly connect the components of the kit.

The kit may comprise a collection channel fluidly connectable to one ofthe first outlet and the second outlet of the microfluidic separationchannel. The kit may comprise a collection vessel fluidly connectable tothe collection channel. The kit may comprise a collection channelfluidly connectable to the first outlet and configured to recycle targetparticle enriched fluid or target particle depleted fluid to themicrofluidic separation channel or biofluid. The kit may comprise anintraluminal line fluidly connectable to one of the microfluidicseparation channel and the first or the second outlet. The kit maycomprise more than one microfluidic separation channel fluidlyconnectable to the source of the biofluid in parallel or in series. Thekit may comprise one or more sensors or control modules connectable tothe microfluidic separation channel.

The kit may include instructions to collect a biofluid, pretreat thebiofluid by introducing a predetermined volume of additive into thesource of the biofluid, flow the pretreated biofluid through themicrofluidic separation channel, and apply acoustic energy to theseparation channel. The kit may provide instructions to introduce atarget particle into the biofluid to provide a therapeutic treatment toat least one component of the biofluid. The kit may further provideinstructions to detach non-target particles bound to the targetparticles, for example, before introducing an additive into thebiofluid.

In some embodiments, the kit provides instructions to introduce theadditive to alter or regulate the density of the biofluid orconcentration of the target particles or non-target particles. The kitmay comprise instructions to introduce a predetermined volume of theadditive to control a desired density of the pretreated biofluid, aspreviously discussed herein. For instance, the kit may compriseinstructions to introduce the additive to regulate the density of thebiofluid to a substantially match a density of the target particles ornon-target particles. The kit may further comprise instructions tocontrol the power, voltage, or frequency of the acoustic transducer toalter or regulate the HCT %, concentration of target particles orconcentration of non-target particles in the output suspension, aspreviously discussed herein. For instance, the kit may compriseinstructions to regulate the output suspension HCT % to be less thanabout 1%. The kit may comprise instructions to perform any step orcollection of steps from the method of separating target particles fromnon-target particles.

The function and advantages of the embodiments discussed above and otherembodiments of the invention can be further understood from thedescription of the figures below, which further illustrate the benefitsand/or advantages of the one or more systems and techniques of theinvention but do not exemplify the full scope of the invention.

As shown in the exemplary concept schematic drawing of FIG. 1, abiofluid comprising target particles 18 and non-target particles 16 and20 is flowed through microfluidic separation channel 28, through theinlet 10. Acoustic energy is applied to the separation channel 28 withinthe illustrated dotted line rectangle. Acoustic energy may be applied byattaching a piezoelectric transducer (not shown) to one wall of theseparation channel. Target particles 18 accumulate within primary steam32 and exit the separation channel 28 through first outlet 14. Targetparticle enriched fluid exits the first outlet 14. Non-target particles16 and 20 accumulate within secondary steam 30 and exit the separationchannel through second outlet 12. The non-target particles 18 and 20exit second outlet 12 in a non-target particle enriched fluid. In someembodiments, the target particle enriched fluid within the primary steam32 is collected. In some embodiments, the non-target particle enrichedfluid within the secondary steam 30 is collected.

Similarly, as shown in the exemplary concept schematic drawing of FIG.2, the biofluid comprising target particles 18 and non-target particles16 and 20 is flowed through the microfluidic separation channel 28through inlet 10. In the embodiment exemplified in FIG. 2, targetparticles 18 essentially accumulate within two primary streams, 34 and38, at the periphery of the separation channel 28, upon being subjectedto the acoustic energy. Non-target particles 16 and 20 essentiallyaccumulate within the central secondary stream 36. The primary streams34 and 38 (target particle enriched fluid) exit the separation channel28 through peripheral first outlets 22 and 26, while the secondarystream 36 (waste fluid) exits the separation channel 28 through secondoutlet 24. In this exemplary embodiment, non-target particles 16 and 20are more susceptible to the acoustic energy, so they travel rapidly tothe central region (secondary stream 36) of the separation channel 28,while the target particles 18 experience a weaker force from theacoustic energy and remain in the peripheral region of the separationchannel 28 (primary streams 34 and 38).

FIG. 3 and FIG. 4 are microscopic images of the downstream end of amicrofluidic separation channel. In FIG. 3, the microfluidic separationchannel is receiving no acoustic energy. As shown in the image, ahomogeneous biofluid suspension is flowing through the channel with noseparation. In FIG. 4, the microfluidic separation channel is receivingacoustic energy. Non-target particles, shown as the darker shade, can beseen traveling through the center stream, while target particles (notindividually visible in the images) travel through the outer streams.The separation as seen in FIG. 4 is much greater than that seen in FIG.3.

As shown in FIG. 5, according to certain embodiments, a system formicrofluidic separation of target particles and non-target particles ina biofluid may comprise a source of a biofluid 110, a source of anadditive 120, and a microfluidic separation channel 140 coupled to anacoustic transducer 240. The system may further comprise a sensor 180configured to measure a parameter of an input biofluid and a sensor 360configured to measure a parameter of a primary stream. In someembodiments, a sensor (not shown) is configured to measure a parameterof the secondary stream. The sensors may be electrically connected tocontrol modules 340 and 160, such that control module 340 is configuredto alter or regulate an input parameter of the acoustic transducer 240and the control module 160 is configured to introduce a predeterminedvolume of the additive into the biofluid.

The system may further comprise intraluminal line 260 fluidly connectedto donor subject 280 and second intraluminal line 300 fluidly connectedto recipient subject 320. Recipient subject 320 and donor subject 280may be the same subject. The microfluidic separation channel 140 mayseparate pretreated biofluid into a primary stream and a secondarystream, such that the primary stream comprising target particles (targetparticle enriched fluid) is directed to primary stream collectionchannel 200 and the secondary stream comprising non-target particles(target particle depleted fluid) is directed to secondary streamcollection channel 220. The secondary stream 220 may be recycled back tothe source of the biofluid 110 through recycle line 380 or may bepost-treated in post-treatment chamber 400. In some embodiments, thepost-treatment chamber 400 is fluidly connected to the intraluminal line300. The primary stream 200 may be collected in collection vessel 420.The system may further comprise a source of a second fluid 460 fluidlyconnected to the microfluidic separation channel 140.

Turning to FIG. 6, the system for microfluidic separation of targetparticles and non-target particles in a biofluid may further comprisetwo or more microfluidic separation channels 140. In the embodiment asshown, each microfluidic separation channel 140 is coupled to anacoustic transducer 240, however the system may comprise one acoustictransducer 240 coupled to more than one microfluidic separation channel140. The two or more microfluidic separation channels 140 may be fluidlyconnected to a manifold 440, which may be fluidly or electricallyconnected to a sensor 500. The manifold 440 may be configured distributethe biofluid to the microfluidic separation channels 140 in response toa measurement received from the sensor 500 of an input biofluid loadupstream of the biofluid source 110. In some embodiments, the systemcomprises a collection channel 200 downstream from the microfluidicseparation channels 140 configured to collect the primary stream orsecondary stream from the microfluidic separation channels 140. Thesystem may further comprise a collection vessel 480 downstream from thecollection channel 200.

The system may further comprise a source of a target particle 600, atarget particle processing chamber 620, and a recycle line 640configured to return target particles to the biofluid, as shown in theexemplary schematic diagram of FIG. 7.

As shown in exemplary concept schematic drawing of FIG. 8, a secondfluid 42 may be flowed through the microfluidic separation channel 28with pretreated biofluid 40, in essentially parallel flow. The secondfluid 42 enters the microfluidic separation channel 28 through centralinlet 46, while pretreated biofluid 40 enters the microfluidicseparation channel 28 through peripheral inlets 44 and 48. The secondfluid 42 does not comprise particles or cells as it enters theseparation channel 28, and may be an inert fluid. Non-target particles16 and 20 are driven towards the center stream by the applied acousticenergy, and exit the separation channel through waste outlet 24. Targetparticles 18 are essentially buoyant within the microfluidic separationchannel 28, and are not driven to the central stream. The estimatedrecovery in the exemplary embodiment of FIG. 8 is calculated to be about70%. Comparatively, the estimated recovery in an embodiment withoutintroducing a second fluid, such as the one exemplified in FIG. 2, isabout 65%.

EXAMPLES Example 1 Prophetic Example of Acoustic Separation forPurification of Target Particles

Separation of target particles, for example cell culture carrierparticles in a biofluid comprising mesenchymal stem cells, may beperformed with a microfluidic separation channel. The mesenchymal stemcells may be collected from a human subject. Cell culture carrierparticles are introduced into biofluid to culture the stem cells. Aftercell culture period, the cultured stem cells may be detached from thecell culture carrier particles by reaction with a biochemical additive.The biofluid comprising cultured stem cells and carrier particles mustbe processed to separate the carrier particles before the cultured stemcells may be introduced into a human subject.

The biofluid comprising stem cells and carrier particles is flowedthrough a microfluidic separation channel, for example, at a residencetime of about 1 second. Ultrasonic waves may be applied to the channelto oscillate a portion of the channel having a cross section on thescale of the ultrasonic wavelength (˜1 mm). The acoustic energy on thechannel may be applied to drive the carrier particles toward an axialcenter stream.

The carrier particles and cultured cells may experience differentacoustic forces. For example, the carrier particles may experience aweaker force than the cultured stem cells and other cells or particleswithin the biofluid. As the biofluid is flowed through the separationchannel, the target particle enriched fluid, here the fluid comprisingcarrier particles, is accumulated along primary streams at the outsideof the channel. The stem cell enriched fluid (comprising the non-targetparticles) is accumulated along secondary streams and separated by abranching in the channel. The stem cell enriched fluid is collected andanalyzed. The carrier particle enriched fluid may be collected andwashed for further use culturing cells.

Accordingly, target particle carrier particles may be separated fromcultured stem cells and other non-target particles, according to themethods described herein.

Example 2 Prophetic Example of Acoustic Separation with an Additive

Biofluid comprising target particles and non-target particles may besubjected to acoustic energy, generally as described above. Prior toflowing the biofluid through a microfluidic separation channel, samplesmay be pretreated by diluting with an additive, for instance with adensity gradient medium at diluent densities ranging between about 1.00and 1.15 (g/mL). The results may be measured in separation ratio, aquantitative measurement of the ratio of target particles in the product(separation efficiency).

The separation Ratio for any subpopulation x, where “side” is theprimary stream and “center” is the secondary stream.

${SR}_{x} = \frac{n_{x,{side}}}{n_{x,{side}} + n_{x,{center}}}$

The fraction of the stream out the side channel (primary stream), alsoreferred to as the flow split:

${FR}_{side} = \frac{V_{side}}{V_{{center}\;}}$

The additive may provide efficient separation of target particles fromother non-target particles. A maximum separation of target particlesfrom other non-target particles may be effectuated near the density ofthe target particles.

Accordingly, target particle separation from non-target particles in abiofluid can be performed with superior results by pretreating thebiofluid with an additive, such as a density gradient medium. Withoutwishing to be bound to a particular theory, it is believed particleseparation by pretreatment with additives capable of altering density ofthe biofluid, density of the target particles, density of the non-targetparticles, size of the target particles, size of the non-targetparticles, compressibility of the biofluid, compressibility of thetarget particles, compressibility of the non-target particles,aggregation potential of the target particles, and aggregation potentialof the non-target particles will provide superior results over nopretreatment because the rate at which the particles migrate generallydepends on particle size, density, and compressibility relative to thedensity and compressibility of the suspending biofluid.

Example 3 Prophetic Example of Acoustic Separation with an Additive

Biofluid comprising target particles and non-target particles may besubjected to acoustic energy, generally as described above. Prior toflowing the biofluid through a microfluidic separation channel, samplesmay be pretreated by introducing a cell aggregator. For example, Ficoll™PM 300 cell media (GE Healthcare, Chicago, Ill.), a long-chainpolysaccharide may be introduced into the biofluid.

The samples pretreated with a cell aggregator may exhibit betternon-target particle removal (for example, erythrocytes or cultured stemcells)than the samples that were not pretreated. Both the cellaggregator samples and the density gradient medium samples, as describedabove, may exhibit improved non-target particle removal and targetparticle recovery than control samples pretreated with PBS alone.

It is expected that, pretreating the biofluid with a cell aggregatorwill provide superior non-target particle removal, but inferior targetparticle recovery, than pretreating the biofluid with a density gradientmedium. Furthermore, non-target particle separation from targetparticles in a biofluid can be performed with superior results bypretreating the biofluid with a density gradient medium and a cellaggregator, as compared to pretreating the biofluid with either additiveindividually or PBS alone.

Those skilled in the art should appreciate that the parameters andconfigurations described herein are exemplary and that actual parametersand/or configurations will depend on the specific application in whichthe disclosed methods and materials are used. Those skilled in the artshould also recognize or be able to ascertain, using no more thanroutine experimentation, equivalents to the specific embodimentsdisclosed. For example, those skilled in the art may recognize that themethod, and components thereof, according to the present disclosure mayfurther comprise a network or systems or be a component of a system formicrofluidic particle separation. It is therefore to be understood thatthe embodiments described herein are presented by way of example onlyand that, within the scope of the appended claims and equivalentsthereto; the disclosed embodiments may be practiced otherwise than asspecifically described. The present systems and methods are directed toeach individual feature, system, or method described herein. Inaddition, any combination of two or more such features, systems, ormethods, if such features, systems, or methods are not mutuallyinconsistent, is included within the scope of the present disclosure.The steps of the methods disclosed herein may be performed in the orderillustrated or in alternate orders and the methods may includeadditional or alternative acts or may be performed with one or more ofthe illustrated acts omitted.

Further, it is to be appreciated that various alterations,modifications, and improvements will readily occur to those skilled inthe art. Such alterations, modifications, and improvements are intendedto be part of this disclosure, and are intended to be within the spiritand scope of the disclosure. In other instances, an existing facilitymay be modified to utilize or incorporate any one or more aspects of themethods and systems described herein. Thus, in some instances, thesystems may involve microfluidic particle separation. Accordingly theforegoing description and figures are by way of example only. Furtherthe depictions in the figures do not limit the disclosures to theparticularly illustrated representations.

The phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. As used herein, theterm “plurality” refers to two or more items or components. The terms“comprising,” “including,” “carrying,” “having,” “containing,” and“involving,” whether in the written description or the claims and thelike, are open-ended terms, i.e., to mean “including but not limitedto.” Thus, the use of such terms is meant to encompass the items listedthereafter, and equivalents thereof, as well as additional items. Onlythe transitional phrases “consisting of‘and “consisting essentially of,”are closed or semi-closed transitional phrases, respectively, withrespect to the claims. Use of ordinal terms such as “first,” “second,”“third,” and the like in the claims to modify a claim element does notby itself connote any priority, precedence, or order of one claimelement over another or the temporal order in which acts of a method areperformed, but are used merely as labels to distinguish one claimelement having a certain name from another element having a same name(but for use of the ordinal term) to distinguish the claim elements.

While exemplary embodiments of the disclosure have been disclosed, manymodifications, additions, and deletions may be made therein withoutdeparting from the spirit and scope of the disclosure and itsequivalents, as set forth in the following claims.

What is claimed is:
 1. A method of separating target particles fromnon-target particles in a biofluid, the target particles having beenintroduced into the biofluid, the method comprising: pretreating thebiofluid by introducing an additive to alter at least one of size of thetarget particles, size of the non-target particles, compressibility ofthe biofluid, compressibility of the target particles, compressibilityof the non-target particles, aggregation potential of the targetparticles, and aggregation potential of the non-target particles;flowing the pretreated biofluid into an inlet of a microfluidicseparation channel; and applying acoustic energy to the microfluidicseparation channel, such that the target particles accumulate within atleast one primary stream along the separation channel and the non-targetparticles accumulate within at least one secondary stream along theseparation channel.
 2. The method of claim 1, further comprisingcollecting the at least one primary stream comprising the targetparticles.
 3. The method of claim 2, further comprising separatelycollecting the at least one secondary stream comprising the non-targetparticles.
 4. The method of claim 1, further comprising selecting theadditive from the group consisting of a cell aggregator, deionizedwater, a detergent, a surfactant, a solution to regulate salinity of thebiofluid, a solution to regulate tonicity of the biofluid, a solution toregulate viscosity of the biofluid, a solution to regulate osmolarity ofthe biofluid, a solution to regulate ion concentration of the biofluid,and combinations thereof.
 5. The method of claim 4, further comprisingselecting the cell aggregator to be a long-chain polysaccharide.
 6. Themethod of claim 5, wherein the cell aggregator comprises a long-chainpolysaccharide having a molecular weight between 100 and 500 kD.
 7. Themethod of claim 5, wherein the cell aggregator comprises a long-chainpolysaccharide present at a concentration of between about 0.5% (w/v)and about 25% (w/v).
 8. The method of claim 4, further comprisingselecting the cell aggregator to be a solution comprising antibodiesthat bind and aggregate non-target particles.
 9. The method of claim 4,further comprising selecting the cell aggregator to be a plateletactivator or a cell adhesion molecule.
 10. The method of claim 1,further comprising introducing an additive to alter at least one ofdensity of the biofluid, density of the target particles, and density ofthe non-target particles.
 11. The method of claim 10, further comprisingselecting the additive from the group consisting of a density gradientmedium, a density additive, and combinations thereof.
 12. The method ofclaim 11, further comprising selecting the density additive to be anonionic iodinated compound.
 13. The method of claim 10, furthercomprising introducing the additive to regulate the density of thebiofluid to substantially match a density of the target particles. 14.The method of claim 13, further comprising introducing the additive toregulate the density of the biofluid to a density of between about 1.00g/mL and about 1.15 g/mL.
 15. The method of claim 10, comprisingintroducing an additive to alter density of the biofluid and aggregationpotential of the non-target particles.
 16. The method of claim 1,further comprising selecting the biofluid from blood buffy coat,leukapheresis product, peripheral blood, whole blood, lymph fluid,synovial fluid, spinal fluid, bone marrow, ascities fluid, andcombinations or subcomponents thereof.
 17. The method of claim 1,further comprising selecting the target particles to be syntheticparticles selected from the group consisting of carrier particles,capture particles, enrichment particles, delivery particles, subclassesthereof, and combinations thereof.
 18. The method of claim 1, furthercomprising selecting the target particles to be cell culture carrierparticles.
 19. The method of claim 1, further comprising obtaining thebiofluid from a donor subject.
 20. The method of claim 1, furthercomprising post-treating the at least one secondary stream.
 21. Themethod of claim 20, further comprising collecting the post-treatedsecondary stream.
 22. The method of claim 20, further comprisingintroducing the post-treated secondary stream into a recipient subject.23. The method of claim 1, wherein the biofluid comprises non-targetparticles bound to the target particles, the method further comprisingtreating the biofluid to unbind the non-target particles for separation.24. The method of claim 1, wherein the therapeutic treatment provided bythe target particle prior to pretreating the biofluid is selected fromthe group consisting of delivering a therapeutic moiety, capturing atherapeutic moiety, capturing a toxin, capturing a cell type, capturinga synthetic particle, culturing a cell type, and combinations thereof.25. The method of claim 1, further comprising flowing a second fluidadjacent to the biofluid into an inlet of the microfluidic separationchannel, such that the biofluid and the second fluid flow insubstantially parallel, substantially laminar flow.
 26. The method ofclaim 1, further comprising flowing the pretreated biofluid into theinlet of the microfluidic separation channel at a flow rate of betweenabout 0.03 mL/min to about 0.5 mL/min.
 27. The method of claim 1,comprising introducing an additive to regulate the aggregation potentialof the non-target particles, the non-target particles comprisingerythrocytes.
 28. The method of claim 1, comprising introducing anadditive to regulate the aggregation potential of the non-targetparticles, the non-target particles comprising platelets.
 29. The methodof claim 1, wherein at least one of the non-target particles comprise atleast one of live cells, frozen cells, preserved cells, and cells grownin a cell culture.
 30. A system for microfluidic particle separationconfigured to separate target particles from non-target particles in abiofluid, the system comprising: at least one microfluidic separationchannel comprising at least one inlet, a first outlet, and a secondoutlet; a source of the biofluid in fluid communication with the atleast one inlet of the at least one microfluidic separation channel; asource of an additive in fluid communication with the source of thebiofluid, configured to introduce at least one additive into thebiofluid, the additive capable of altering at least one of size of thetarget particles, size of the non-target particles, compressibility ofthe biofluid, compressibility of the target particles, compressibilityof the non-target particles, aggregation potential of the targetparticles, and aggregation potential of the non-target particles; and atleast one acoustic transducer coupled to a wall of the at least onemicrofluidic separation channel.
 31. The system of claim 30, wherein theat least one acoustic transducer is positioned to apply a standingacoustic wave transverse to the microfluidic separation channel.
 32. Thesystem of claim 30, further comprising at least two microfluidicseparation channels connected in parallel and a manifold configured todistribute the biofluid to the at least two microfluidic separationchannels.
 33. The system of claim 32, further comprising at least onesensor configured to measure an input biofluid load on the system. 34.The system of claim 33, wherein the manifold is in electricalcommunication with the at least one sensor, configured to distribute thebiofluid to the at least two microfluidic separation channels inresponse to a measurement of the input biofluid load on the system. 35.The system of claim 30, wherein the additive is further capable ofaltering at least one of density of the biofluid, density of the targetparticles, and density of the non-target particles.
 36. The system ofclaim 35, further comprising at least one sensor configured to measureat least one of density of the biofluid and concentration of targetparticles or non-target particles.
 37. The system of claim 36, furthercomprising a control module in electrical communication with the atleast one sensor and the source of the additive, configured to introducea predetermined volume of the additive into the biofluid in response toa measurement of at least one of the density of the biofluid and theconcentration the target particles or the non-target particles in thebiofluid.
 38. The system of claim 37, wherein the predetermined volumeis determined to regulate the density of the biofluid to substantiallymatch a density of the target particles.
 39. The system of claim 38,wherein the predetermined volume is determined to regulate the densityof the biofluid to a density of between about 1.00 g/mL and about 1.15g/mL.
 40. The system of claim 30, further comprising at least one sensorconfigured to measure at least one parameter of an output suspension.41. The system of claim 40, further comprising at least one sensorconfigured to measure at least one of hematocrit (HCT %) of the outputsuspension and concentration of the target particles or the non-targetparticles in the output suspension.
 42. The system of claim 40, furthercomprising a control module in electrical communication with the atleast one sensor and the acoustic transducer, configured to regulate atleast one of power, voltage, and frequency delivered to the acoustictransducer in response to a measurement of the at least one parameter inthe output suspension.
 43. The system of claim 42, wherein the controlmodule is configured to regulate the HCT % of the output suspension toless than about 1%.
 44. The system of claim 30, further comprising atleast one sensor configured to measure at least one of HCT % of thebiofluid and concentration of target particles or non-target particles.45. The system of claim 44, further comprising a control module inelectrical communication with the at least one sensor and the source ofthe additive, configured to introduce a predetermined volume of theadditive into the biofluid in response to a measurement of at least oneof the HCT % of the biofluid and the concentration the target particlesor the non-target particles in the biofluid.
 46. The system of claim 45,wherein the predetermined volume of the additive is determined toregulate the HCT % of the biofluid to less than about 10%.
 47. Thesystem of claim 30, further comprising a source of a second fluid influid communication with the at least one inlet of the at least onemicrofluidic separation channel, configured to introduce the secondfluid into the biofluid, such that the biofluid and the second fluidflow in substantially parallel, substantially laminar flow.
 48. Thesystem of claim 30, further comprising a first collection channel influid communication with the first outlet of the microfluidic separationchannel and a second collection channel in fluid communication with thesecond outlet of the microfluidic separation channel.
 49. The system ofclaim 48, wherein the second collection channel comprises a recycle linein fluid communication with the source of the biofluid, configured torecycle target particle depleted fluid from the second outlet to thesource of the biofluid.
 50. The system of claim 48, wherein the first orsecond collection channel is in fluid communication with a collectionvessel.
 51. The system of claim 30, connectable to an intraluminal linein fluid communication with a donor subject and the source of thebiofluid, configured to extract biofluid from the donor subject anddeliver the biofluid to the source of the biofluid.
 52. The system ofclaim 30, connectable to an intraluminal line in fluid communicationwith one of the first and the second outlet of the separation channeland a recipient subject, configured to deliver output suspension to therecipient subject.
 53. The system of claim 30, wherein the microfluidicseparation channel is formed from a thermoplastic material.
 54. A kitfor microfluidic particle separation comprising: at least onemicrofluidic separation channel configured to separate target particlesfrom non-target particles in a biofluid, the separation channelcomprising at least one inlet, a first outlet, and a second outlet, theseparation channel connected to at least one acoustic transducer; asource of an additive fluidly connectable with the microfluidicseparation channel, the additive capable of altering at least one ofsize of the target particles, size of the non-target particles,compressibility of the biofluid, compressibility of the targetparticles, compressibility of the non-target particles, aggregationpotential of the target particles, and aggregation potential of thenon-target particles; and instructions to provide a biofluid, pretreatthe biofluid by introducing a predetermined volume of the additive intothe biofluid, flow the pretreated biofluid into the at least one inletof the microfluidic separation channel, and apply acoustic energy to themicrofluidic separation channel.
 55. The kit of claim 54, furthercomprising a collection channel fluidly connectable to one of the firstoutlet and the second outlet.
 56. The kit of claim 55, furthercomprising a collection vessel fluidly connectable to the collectionchannel.
 57. The kit of claim 55, wherein the collection channel isfluidly connectable to the first outlet and configured to recycle targetparticle depleted fluid to the at least one inlet of the microfluidicseparation channel.
 58. The kit of claim 54, wherein the at least onemicrofluidic separation channel is formed of a thermoplastic material.59. The kit of claim 58, wherein the thermoplastic microfluidicseparation channel is disposable.
 60. The kit of claim 54, furthercomprising an intraluminal line fluidly connectable to one of themicrofluidic separation channel and the first outlet.
 61. The kit ofclaim 54, wherein the additive is further capable of altering at leastone of density of the biofluid, density of the target particles, anddensity of the non-target particles.
 62. The kit of claim 61, furthercomprising instructions to introduce the additive to regulate thedensity of the biofluid to substantially match a density of the targetparticles.